| Glioblastoma is the most common and aggressive brain humor and its incidence is3.17/100,000per year. It occupies17%of primary tumors. The5-year and10-year survivalrate of glioblastoma is4.5%and2.7%separately. Although the treatment of glioblastoma isupdated in nursing, surgery, radiotherapy and chemotherapy, its median non-progressive stageand median survival time is still6.7months and14.6months. Although the treatments ofglioblastoma in terms of surgery, radiotherapy and chemotherapy have already been welldeveloped, the prognosis has no considerable progress. Surgery combined with whole brainradiotherapy is still the first choice. There is no other therapy could clearly replace thesurgery and radiotherapy. Besides the infiltrating growth pattern, the terrible prognosis ofglioblastoma also refers to its special immunosuppressive phenotype. In addition,glioblastoma cells are almost resistant to various apoptotic stimulation factors. Changingapoptotic pathways could not only induce gliomagenesis, but also result in resistance toclassical genotoxic therapy. The present treatment cannot control and treat the tumoreffectively so the creative therapy strategy needs to be developed. In the past decades, plentyof work was done to analyze the changes in the molecular pathways of apoptosis ofglioblastoma cells and then to make them more sensitive to therapy induced apoptosis. Someresearches suggested that the under physiological conditions precisely regulated apoptoticcell death could also be seen in glioblastoma, resulting in a survival in glioblastoma cells.Furthermore, critical roles of the apoptotic cascades appeared in tumor cells and then tendedto theraputic interventions. Up to now, however, clinical trials most of which have focused ongrowth factor have investigated little effect. The greatest challenge is still to apply thepreclinical achievemenst to clinical trials in glioblastoma and to avoid side-effects on healthytissues, at the same time to retain the curative efficacy to apoptotic pathways.Lithocholic acid is a kind of bile acid. In the recent researches, low concentrations ofLCA could make BE(2)-m17and SK-n-MCIXC cells sensitive to H2O2-induced death ofapoptotic cells which was regulated by mitochondria. The certain concentrations could make primary cultures of human neurons resist to the death. In BE(2)-m17and SK-n-MCIXC NBcell lines, LCA could bind to the cell surface and make the intracellular cascades start whichcan result in cell death to induce internal and external apoptosis pathways. Recent studiesshow that LCA binding to the surface of BE(2)-m17and SKn-MCIXC cells can induce cellapoptosis to respond to the internal and external apoptotic pathways by means of cascadereactions. TGR5stimulated by LCA could initiate three molecular cascades to start the cellapoptosis of internal and external pathways. In the cultured Lan-1human NB cell line, thepast studies implied different kinds of mechanisms are the basis of the anti-tumor effect ofLCA. Prior studies suggest that the anti-tumor ability of LCA in cultured human NB cells andthe longevity extending ability of LCA in quiescent yeast attribute to LCA’s capacity toregulate mitochondria-limited procedures which are indispensable parts in malignant tumorand aging. The effects of LCA on mitochondrial in cultured NB cell s are partly differentfrom those in quiescent yeast, especially in primary human neuron cultures. In fact, whileLCA strengthens the sensitivity of BE(2)-m17and SKn-MC NB cell lines tomitochondria-confined apoptotic cell death caused by externally added H2O2, it considerablymakes the resistance of quiescent yeast and primary human neuron cultures increase. LCA isof great value on the anti-tumor research because it can induce the neurogenic tumorapoptosis by means of cascade reaction and can protect the normal neurocytes with certainconcentration.Lithocholic acid (LCA) killed glioma cells and spared normal neuronal cells.Anti-glioma mechanism of LCA is unclear for the moment, especially whether it hasregulatory effect on glioblastoma. The prior researches showed that unsaturated aldehydescould activate the apoptosis pathway like Fas/FasL, but reactive oxygen could damage thestructure and function of cells, even cause them to death. Leak of protons likely induced bylipid peroxidation and backed into matrix of mitochondria and limited production of ROSresulting in uncoupling of oxidative phosphorylation. Uncoupling brought about via theleak of protons through downstream lipid peroxidation products. Lipid peroxidation causedfree radical reactions resulting in various kinds of aldehydes products. It is a signalingmolecule of mitochondria. It usually causes mutations to trigger oncogenesis. Therefore to establish the unsaturated aldehydes model can provide the method, bridge and platform forthe researches of the molecular mechanism of LCA’s effect on glioblastoma. We chose theglioblastoma mitochondria to be the object of study and established the lipid peroxidationmodel induced by hydrogen peroxide. We also detected the unsaturated aldehydes under theintervention of LCA and explore its regulatory ability to unsaturated aldehydes in order toilluminate primarily the molecular mechanism of LCA’s anti-glioblastoma by means ofmitochondria. At last we successfully established the bioactive unsaturated aldehydesmitochondria model to apply to the in vitro mechanism research of the regulation of LCA toglioblastoma. The optimum cutoff conditions have been confirmed according to change of thepeaks in495nm,450nm and532nm using lipid peroxidation model via glioma mitochondriaby means of reactions between2-thiobarbituric acid and biological active α, β-unsaturatedaldehydes. The study concluded that mitochondrial modeling in glioma lipid peroxidationwas successfully established and the model conditions were glioma mitochondrialconcentration1.5mg/ml, H2O2concentration0.6mg/ml, duration of action30min and3.0ml46mM TBA. The model observed indicators were changes of the peaks of495nm,450nm and532nm related to aldehydes in lipid peroxidation of glioma mitochondria induced by H2O2.Regulation of LCA on the optimum cutoff peaks of450nm,495nm, and532nm were100μM,duration of action15min and acidic microenvironment according to the newly createdmitochondria model. The suitable concentration of LCA has anti-glioma effects by means ofregulation on changes of peaks in450nm,495nm, and532nm and the glioma mitochondrialmodel conducive to in-depth research.E-2-Butenal (Crotonaldehyde, Scheme2), a hydrophilic α, β-unsaturated aldehyde, isproduced endogenously from mitochondrial lipid peroxidation as well, efficiently absorbed inproduced sites, and then reacted with DNA for the formation of exocyclic DNA adducts.Arnold N. Onyango (2012) reviewed on the basis of some valuable literatures shown thebioactive carbonyl compounds such as E-2-Butenal contributed certainly to lipid oxidationand carcinogen. Its ultraviolet spectrum peak is495nm, so the LCA`s effects on anti-gliomaconcluded by the former study by means of regulation on changes of450nm,495nm, and532nm peaks could be realized by regulating E-2-Butenal. But at present, the molecular level researches on the relationships of LCA, E-2-Butenal and glioblastoma are rare, especially themechanism reports. As is well-known, the interaction characteristics of LCA and E-2-Butenalis of great importance to illuminate the mechanism of LCA to E-2-Butenal. Hence weadopted E-2-Butenal as target, using UV-visible spectra and Raman spectra, to observe thechanges of LCA. On one hand, it is helpful to illuminate the regulatory molecular mechanismof LCA to E-2-Butenal to explore the tunable molecular target of LCA to E-2-Butenal. On theother hand, it could provide fundamental basis for studying the molecular mechanism ofanti-glioblastoma of LCA. In this work, the molecular regulation of lithocholic acid onE-2-Butenal using both UV-visible spectra in the range of400–600nm and Raman spectra inthe range of2000–500cm1were studied. The results of UV-visible spectrum detectiondisplayed that the characteristics of lithocholic acid on E-2-Butenal were gradually decreasedat the main peak of494nm in the concentration-dependent. This indicated that theinterreaction of lithocholic acid and E-2-Butenal according to aldol reaction could explain thespectral phenomenon. The results of Raman spectrum detection revealed that the downtrendof the very strong band of1676cm1and strong band of at1642cm1were obvious. Inconclusion, these results clearly suggested that the molecular targets of down regulation forlithocholic acid on E-2-Butenal were C=O and C=C. The tunable evidences of lithocholicacid on E-2-Butenal will helps to make sure for lithocholic acid as a novel drug to cureglioma related to exposure E-2-Butenal microenvironment. The present study provided thetunable evidences of LCA on E-2-Butenal by UV-visible and Raman detection. The resultsshown that the down regulation of LCA on E-2-Butenal (Crotonaldehyde) were the mainpeak of494nm in UV-visible spectra and the very strong band of1676cm1and strong bandof at1642cm1in Raman spectra. Reduction of E-2-Butenal content was clearly observedusing different concentrations of LCA. With LCA were increased, the contents ofE-2-Butenal were obviously decreased. The regulation and control targets of LCA onE-2-Butenal were C=O and C=C stretching vibrations. The possible molecular mechanismsof LCA on E-2-Butenal were decreasing electronic cloud of the oxygen atom in CHO andthen the CHO was kept inactive state, prevented the molecular polarization of conjugateddouble bonds, and influenced methyl inducing and hyperconjugation effects. These results would help to establish LCA as a novel drug for glioma related to E-2-Butenal. |
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